Method for the detection of egfr mutations in blood samples

ABSTRACT

The present invention refers to the detection of EGFR mutations in a blood (serum/plasma) sample from a subject. The method comprises:
         (i) obtaining the DNA from said sample;   (ii) amplifying the nucleic acid sequence corresponding to a specific region of the EGFR gene by means of PCR using a Protein-Nucleic Acid probe; and   (iii) detecting said mutation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 USC 120 of U.S. patentapplication Ser. No. 12/374,307 filed Oct. 10, 2011, which in turn is aU.S. national phase under the provisions of 35 USC §371 of InternationalApplication No. PCT/EP07/57510 filed Jul. 20, 2007, which in turn claimspriority of European Patent Application No. 06117551.9 filed Jul. 20,2006. The disclosures of such international application and Europeanpriority application are hereby incorporated herein by reference intheir respective entireties, for all purposes.

FIELD OF THE INVENTION

The present invention refers to the detection of EGFR mutations in ablood (serum/plasma) sample from a subject.

BACKGROUND

Lung cancer is the leading cause of cancer-related mortality in both menand women. The prevalence of lung cancer is second only to that ofprostate cancer in men and breast cancer in women. Lung cancer recentlysurpassed heart disease as the leading cause of smoking-relatedmortality. In addition, most patients who develop lung cancer smoke andhave smoking-related damage to the heart and lungs, making aggressivesurgical or multimodality therapies less viable options. Most lungcarcinomas are diagnosed at an advanced stage, conferring a poorprognosis.

Non-small cell lung cancer (NSCLC) accounts for approximately 75% of alllung cancers. NSCLC is a heterogeneous aggregate of histologies. Themost common histologies are epidermoid or squamous carcinoma,adenocarcinoma, and large cell carcinoma.

Several studies have attempted to identify clinical, laboratory, andmolecular markers that may help clinicians and researchers distinguishsubgroups of NSCLC patients. Along these lines, various studies haveshown that epidermal growth factor receptor (EGFR) is over-expressed in40 to 80 percent of non-small cell lung cancers and many otherepithelial cancers.

Aberrant epidermal growth factor receptor (EGFR) signalling is criticalfor limiting sensitivity to anticancer agents and ligand-independenttyrosine kinase activation of EGFR is often caused by EGFR mutations inthe extracellular domain, which has been observed in various tumourtypes, such as glioblastoma multiforme. EGFR signalling is triggered bythe binding of growth factors, such as epidermal growth factor (EGF).Autophosphorylation and transphosphorylation of the receptors throughtheir tyrosine kinase domains leads to the recruitment of downstreameffectors and the activation of proliferative and cell-survival signals.

Two mutations account for approximately 90% of EGFR mutations reportedto date in lung adenocarcinomas. In Caucasian population, the mostcommon mutation type, seen in around 65% of cases with EGFR mutations,is a short in-frame deletion of 9, 12, 15, 18, or 24 nucleotides in exon19. The second most common mutation, seen in about 35% of cases withEGFR mutations, is a point mutation (CTG to CGG) in exon 21 atnucleotide 2573, that results in substitution of leucine by arginine atcodon 858 (L858R) adjacent to the DFG motif in the carboxy-terminal lobein the activation loop of the kinase.

These EGFR mutations are bona fide somatic mutations in NSCLC and havenot been identified in other primary tumour types. Further, EGFRmutations are a strong determinant of tumor response to gefitinib innon-small cell lung cancer (NSCLC). Other much less common mutationshave been described in exons 18, 20, and 21.

So far, screening for these mutations has been based on directsequencing or single-strand conformation polymorphism analysis. Nucleicacid amplification methods (for example, the polymerase chain reaction)allow the detection of small numbers of mutant molecules among abackground of normal ones. While alternative means of detecting smallnumbers of tumor cells (such as flow cytometry) have generally beenlimited to hematological malignancies, nucleic acid amplification assayshave proven both sensitive and specific in identifying malignant cellsand for predicting prognosis following chemotherapy.

Various nucleic acid amplification strategies for detecting smallnumbers of mutant molecules in solid tumor tissue have been developed.For example, one sensitive and specific method identifies mutant rasoncogene DNA on the basis of failure to cleave a restriction site at thecrucial 12th codon (Kahn et al. Rapid and sensitive nonradioactivedetection of mutant K-ras genes via ‘enriched’ PCR amplification.Oncogene. 1991 June; 6(6):1079-83). Similar protocols can be applied todetect any mutated region of DNA in a neoplasm, allowing detection ofother oncogene DNA or tumor-associated DNA. Since mutated DNA can bedetected not only in the primary cancer but in both precursor lesionsand metastatic sites, nucleic acid amplification assays provide a meansof detecting and monitoring cancer both early and late in the course ofdisease.

Other studies have used nucleic acid amplification assays to analyze theperipheral blood of patients with cancer in order to detectintracellular DNA extracted from circulating cancer cells in patients.However, it must be emphasized that these studies attempt to use nucleicacid-based amplification assays to detect extracted intracellular DNAwithin circulating cancer cells. The assay is performed on the cellularfraction of the blood from patients having cancer using the cell pelletor cells within whole blood, and the serum or plasma fraction isconventionally ignored or discarded prior to analysis. Since such anapproach requires the presence of metastatic circulating cancer cells(for non-hematologic tumors), it is of limited clinical use in patientswith early cancers, and it is not useful in the detection ofnon-hematologic non-invasive neoplasms or pre-malignant states.

It is known in the prior art that small but significant amounts ofnormal DNA circulate in the blood of healthy people and this amount hasbeen found to increase in cancer states. The prior art containsdisclosure that mutant oncogene DNA could be detected in peripheralblood plasma or serum of cancer patients. However, these reports havealso been generally limited to patients with advanced cancer or knownneoplastic or proliferative disease. Some authors (Kimura et al., 2006.EGFR Mutation of Tumor and Serum in Gefitinib-Treated Patients withChemotherapy-Naive Non-small Cell Lung Cancer) have described thatmutations at EGFR gene can be detected in serum samples from patientssuffering from NSCLC. Said document describes detection of suchmutations by means of PCR and sequencing using primers flanking saidmutations.

SUMMARY OF THE INVENTION

The present invention refers to a method for the detection of mutationsat the EGFR gene in a blood sample from a subject said methodcomprising:

-   -   (i) obtaining the DNA from said sample;    -   (ii) amplifying the nucleic acid sequence corresponding to a        specific region of the EGFR gene by means of PCR using a        Protein-Nucleic Acid probe; and    -   (iii) detecting said mutation.

Here, the inventors have developed and validated a polymerase chainreaction (PCR)-based assay for the detection of the most common EGFRmutations in plasma/serum samples. This assay offers higher analyticalsensitivity by enhancing amplification of mutant alleles in the samplesto be analysed by means of using a Protein-Nucleic Acid (PNA) probecompared to standard methods, in which primers flanking the mutationsare employed for PCR amplification and further sequencing analysis.Thus, a robust and accessible approach is here provided to the rapididentification of most lung cancer patients who are likely to respond tospecific EGFR inhibitors.

Additionally, while direct analysis of neoplastic tissue is frequentlydifficult or impossible (such as in instances of occult, unrecognizeddisease), the method described above has the advantage of using blood,such as peripheral blood, for the detection of said mutation. Peripheralblood is easily accessible and amenable to nucleic acid-based assays.

DETAILED DESCRIPTION OF THE INVENTION

In order to facilitate the understanding of the present description, themeaning of some terms and expressions in the context of the inventionwill be explained below:

The term “subject”, refers to a male or female human of any age or race.Preferably it includes humans having or suspected of having non-smallcell lung cancer (NSCLC). Diagnostic methods for NSCLC and the clinicaldelineation of NSCLC diagnoses are well known to those of ordinary skillin the medical arts. As examples, methods for identifying subjectssuspected of having NSCLC may include physical examination, subject'sfamily medical history, subject's medical history, lung biopsy, or anumber of imaging technologies such as ultrasonography.

The term “nucleic acid” refers to a multimeric compound comprisingnucleosides or nucleoside analogues which have nitrogenous heterocyclicbases, or base analogues, which are linked by phosphodiester bonds toform a polynucleotide.

The term “DNA” refers to deoxyribonucleic acid. A DNA sequence is adeoxyribonucleic sequence. DNA is a long polymer of nucleotides andencodes the sequence of the amino acid residues in proteins using thegenetic code.

The term “Protein-Nucleic Acid probe” refers to a synthetic DNA analogin which the phosphodiester backbone is replaced by repetitive units ofN-(2-aminoethyl)glycine to which the purine and pyrimidine bases areattached via a methyl carbonyl linker.

In one aspect, the invention refers to a method, herein referred to as“method of the invention”, for the detection of mutations at the EGFRgene in a blood sample from a subject said method comprising:

-   -   (i) obtaining the DNA from said sample;    -   (ii) amplifying the nucleic acid sequence corresponding to a        specific region of the EGFR gene by means of PCR using a        Protein-Nucleic Acid probe; and    -   (iii) detecting said mutation.

The method of the invention can be used to detect any mutation at theEGFR gene. In a particular embodiment of the invention, the mutation atthe EGFR gene to be detected is selected from the group consisting of:ELREA deletions at the exon 19, the L858R mutation at the exon 21 andthe T790M mutation at exon 21.

Samples

Illustrative, non limitative, examples of samples from which nucleicacids can be extracted and analysed using the method of the inventioninclude, but are not limited to, both normal and cancerous blood (serumor plasma) and other body fluids containing nucleic acids that can bedetected.

In order to carry out the method of the invention, a sample is obtainedfrom the subject under study. In a particular embodiment, the sample isa blood sample. Samples can be obtained from subjects previouslydiagnosed or not with NSCLC, or from subjects who are receiving or havepreviously received anti-NSCLC treatment. In an embodiment, the sampleis a sample from a subject having normal lung function tissue, i.e., asubject with no evidence of NSCLC.

In the practice of the invention blood is drawn by standard methods intoa collection tube, preferably comprising siliconized glass, eitherwithout anticoagulant for preparation of serum or with EDTA, heparin, orsimilar anticoagulants, most preferably EDTA, for preparation of plasma.Plasma may optionally be subsequently converted to serum by incubationof the anticoagulated plasma with an equal volume of calcium chloride at37° C. for a brief period, most preferably for 1-3 minutes, untilclotting takes place. The clot may then be pelleted by a briefcentrifugation and the deproteinized plasma removed to another tube.Alternatively, the centrifugation may be omitted. Serum can also beobtained using clot activator tubes.

DNA Amplification

In a particular embodiment of the invention, the serum or plasma may beutilized directly for identification of the mutant DNA. In anotherparticular embodiment, nucleic acid is extracted from plasma or serum asan initial step of the invention. In such cases, the total DNA extractedfrom said samples would represent the working material suitable forsubsequent amplification.

Once the sample has been obtained, amplification of nucleic acid iscarried out. In a particular embodiment, the amplification of the DNA iscarried out by means of PCR. The general principles and conditions foramplification and detection of nucleic acids, such as using PCR, arewell known for the skilled person in the art. In particular, thePolymerase Chain Reaction (PCR) carried out by the method of the presentinvention uses appropriate and specific oligonucleotide primers oramplification oligonucleotides to specifically amplify the EGFR targetsequences. Illustrative, non limitative, examples of such amplificationoligonucleotides include the sequences of SEQ ID NO: 1 and SEQ ID NO: 2.The terms “oligonucleotide primers” or “amplification oligonucleotides”are herein used indistinguishably and refer to a polymeric nucleic acidhaving generally less than 1,000 residues, including those in a sizerange having a lower limit of about 2 to 5 residues and an upper limitof about 500 to 900 residues. In preferred embodiments, oligonucleotideprimers are in a size range having a lower limit of about 5 to about 15residues and an upper limit of about 100 to 200 residues. Morepreferably, oligonucleotide primers of the present invention are in asize range having a lower limit of about 10 to about 15 residues and anupper limit of about 17 to 100 residues. Although oligonucleotideprimers may be purified from naturally occurring nucleic acids, they aregenerally synthesized using any of a variety of well known enzymatic orchemical methods. In a particular embodiment of the invention, sucholigonucleotide primers enable the specific amplification of the DNAfragments corresponding to the deletion of specific nucleotides in theexon 19 at the EGFR gene.

Thus, in a particular embodiment, the method of the invention can beused for the detection of ELREA deletions at the exon 19. In a preferredembodiment, the present invention refers to a method for the detectionof 9, 12, 15, 18, or 24 nucleotides deletions in the exon 19 at the EGFRgene.

In another particular embodiment, the method of the invention can beused for the detection of the L858R mutation at the exon 21 of the EGFRgene. In another embodiment, the method of the invention can be used forthe detection of the T790M mutations in exon 21 of the EGFR gene.

The term “amplification oligonucleotide” refers to an oligonucleotidethat hybridizes to a target nucleic acid, or its complement, andparticipates in a nucleic acid amplification reaction. Amplificationoligonucleotides include primers and promoter-primers in which the 3’end of the oligonucleotide is extended enzymatically using anothernucleic acid strand as the template. In some embodiments, anamplification oligonucleotide contains at least about 10 contiguousbases, and more preferably about 12 contiguous bases, that arecomplementary to a region of the target sequence (or its complementarystrand). Target-binding bases are preferably at least about 80%, andmore preferably about 90% to 100% complementary to the sequence to whichit binds. An amplification oligonucleotide is preferably about 10 toabout 60 bases long and may include modified nucleotides or baseanalogues.

The terms “amplify” or “amplification” refer to a procedure to producemultiple copies of a target nucleic acid sequence or its complement orfragments thereof (i.e., the amplified product may contain less than thecomplete target sequence). For example, fragments may be produced byamplifying a portion of the target nucleic acid by using anamplification oligonucleotide which hybridizes to, and initiatespolymerization from, an internal position of the target nucleic acid.Known amplification methods include, for example, polymerase chainreaction (PCR) amplification, replicase-mediated amplification, ligasechain reaction (LCR) amplification, strand-displacement amplification(SDA) and transcription-associated or transcription-mediatedamplification (TMA). PCR amplification uses DNA polymerase, primers foropposite strands and thermal cycling to synthesize multiple copies ofDNA or cDNA. Replicase-mediated amplification uses QB-replicase toamplify RNA sequences. LCR amplification uses at least four differentoligonucleotides to amplify complementary strands of a target by usingcycles of hybridization, ligation, and denaturation. SDA uses a primerthat contains a recognition site for a restriction endonuclease and anendonuclease that nicks one strand of a hemimodified DNA duplex thatincludes the target sequence, followed by a series of primer extensionand strand displacement steps. An isothermal strand-displacementamplification method that does not rely on endonuclease nicking is alsoknown. Transcription-associated or transcription-mediated amplificationuses a primer that includes a promoter sequence and an RNA polymerasespecific for the promoter to produce multiple transcripts from a targetsequence, thus amplifying the target sequence.

Preferred embodiments of the present invention amplify the EGFR targetsequences using the present amplification oligonucleotides in apolymerase chain reaction (PCR) amplification. One skilled in the artwill appreciate that these amplification oligonucleotides can readily beused in other methods of nucleic acid amplification that usespolymerase-mediated primer extension.

In the amplifying step of the method of the invention, the nucleic acidsequence corresponding to a specific region of the EGFR gene isamplified by means of PCR using a Protein-Nucleic Acid (PNA) probe. PNAprobes are nucleic acid analogs in which the sugar phosphate backbone ofa natural nucleic acid has been replaced by a synthetic peptidebackbone, usually formed from N-(2-aminoethyl)-glycine units, resultingin an achiral and uncharged mimic This new molecule is chemically stableand resistant to hydrolytic (enzymatic) cleavage and thus not expectedto be degraded inside a living cell. Despite all these variations fromnatural nucleic acids, PNA is still capable of sequence-specific bindingto DNA as well as RNA obeying the Watson-Crick hydrogen bonding rules.Its hybrid complexes exhibit extraordinary thermal stability and displayunique ionic strength properties. In many applications, PNA probes arepreferred to nucleic acid probes because, unlike nucleic acid/nucleicacid duplexes which are destabilized under conditions of low salt,PNA/nucleic acid duplexes are formed and remain stable under conditionsof very low salt. Those of ordinary skill in the art of nucleic acidhybridization will recognize that factors commonly used to impose orcontrol stringency of hybridization include formamide concentration (orother chemical denaturant reagent), salt concentration (i.e., ionicstrength), hybridization temperature, detergent concentration, pH andthe presence or absence of chaotropes. Optimal stringency for aprobe/target sequence combination is often found by the well knowntechnique of fixing several of the aforementioned stringency factors andthen determining the effect of varying a single stringency factor. Thesame stringency factors can be modulated to thereby control thestringency of hybridization of a PNA to a nucleic acid, except that thehybridization of a PNA is fairly independent of ionic strength. Optimalstringency for an assay may be experimentally determined by examinationof each stringency factor until the desired degree of discrimination isachieved.

PNA oligomers can be prepared following standard solid-phase synthesisprotocols for peptides (Merrifield, B. 1986. Solid-phase synthesis.Science 232, 341-347) using, for example, a (methyl-benzhydryl)aminepolystyrene resin as the solid support. PNAs may contain a chimericarchitecture, such as a PNA/DNA chimera, where a PNA oligomer is fusedto a DNA oligomer.

Clinical samples contain DNA molecules with the wild-type allele inaddition to DNA molecules with the mutant allele. So, under normalconditions, it is difficult to detect EGFR mutations (mutant allele) ina large background of wild-type EGFR genes (wild-type allele). In aparticular case, the PNA probe utilized by the inventors is capable ofspecifically recognize and hybridize with the wild-type EGFR sequence.As an illustrative, non limitative example, the PNA probe to be used forcarrying out the method of the present invention comprises the PNA probedescribed as the SEQ ID NO:3 in the Example accompanying the presentinvention. Such probe is added to the PCR reaction mix thus inhibitingamplification of the wild-type allele and favouring amplification of themutant allele present in the sample, i.e. EGFR mutant, facilitating itsposterior detection. Those of ordinary skill in the art will appreciatethat a suitable PNA probe do not need to have exactly these probingnucleic acid sequences to be operative but often modified according tothe particular assay conditions. For example, shorter PNA probes can beprepared by truncation of the nucleic acid sequence if the stability ofthe hybrid needs to be modified to thereby lower the Tm and/or adjustfor stringency. Similarly, the nucleic acid sequence may be truncated atone end and extended at the other end as long as the discriminatingnucleic acid sequence remains within the sequence of the PNA probe. Suchvariations of the probing nucleic acid sequences within the parametersdescribed herein are considered to be embodiments of this invention.

As it can be observed in the Example 1 of the present invention, theconditions of the polymerase chain reaction using such PNA probe appliedin the method of the present invention are such that only 40 cycles ofamplification are sufficient for the obtainment of a precise PCRamplification product comprising a 120 by genomic fragment including themutation of interest of exon 19 at the EGFR gene.

The general conditions for the PCR of the method of the presentinvention are as illustrated in the Example 1 of the present invention.In this example, the DNA used for the PCR amplification reaction is fromplasma/serum samples.

Detection of DNA Mutation

Many methods for detecting and analysing the PCR amplification productshave been previously disclosed. Particularly, detection of DNA sequencemutants may proceed by any of a number of methods known to those skilledin the art (Kilger et al., 1997, Nucleic Acids Res. 25: 2032-4). In aparticular embodiment of the invention, the detecting step of the methodof the invention is carried out by means of nucleic acid sequencing.Illustrative, non limitative, examples of nucleic acid sequencingmethods are cycle sequencing (Sarkar et al., 1995, Nucleic Acids Res.23: 1269-70) or direct dideoxynucleotide sequencing, in which some orthe entire DNA of interest that has been harvested from the sample isused as a template for sequencing reactions. An oligonucleotide primeror set of primers specific to the gene or DNA of interest is used instandard sequencing reactions. Other methods of DNA sequencing, such assequencing by hybridization, sequencing using a “chip” containing manyoligonucleotides for hybridization (as, for example, those produced byAffymetrix Corp.; Ramsay et al., 1998, Nature Biotechnology 16: 40-44;Marshall et al., 1998, Nature Biotechnology 16: 27-31), sequencing byHPLC (DeDionisio et al., 1996, J Chromatogr A 735: 191-208), andmodifications of DNA sequencing strategies such as multiplexallele-specific diagnostic assay (MASDA; Shuber et al., 1997, Hum.Molec. Genet. 6: 337-47), dideoxy fingerprinting (Sarkar et al., 1992,Genomics 13: 441-3; Martincic et al., 1996, Oncogene 13: 2039-44), andfluorogenic probe-based PCR methods (such as Taqman; Perkin-Elmer Corp.;Heid et al., 1996, Genome Res. 6: 986-94) and cleavase-based methods maybe used.

Alternatively, amplification can be carried out using primers that areappropriately labelled, and the amplified primer extension products canbe detected using procedures and equipment for detection of the label.Preferably probes of this invention are labeled with at least onedetectable moiety, wherein the detectable moiety or moieties areselected from the group consisting of: a conjugate, a branched detectionsystem, a chromophore, a fluorophore, a spin label, a radioisotope, anenzyme, a hapten, an acridinium ester and a luminescent compound. As anillustrative, non limitative, example, in the method of the presentinvention the primers used can labelled with a fluorophore. Moreparticularly, the reverse primer of the method of the present inventionis labelled with the 6-FAM fluorophore at its 5′ end. This fluorophoreemits fluorescence with a peak wavelength of 522 nm The PCR can becarried out using one of the primers labelled with, for example, eitherFAM, HEX, VIC or NED dyes.

In a preferred embodiment of the invention, the posterior detection andanalysis of the DNA amplified with the method of the invention iscarried out by the GeneScan technique as it is illustrated in theExample accompanying the present invention. Thus, as an illustrative,non limitative, example for carrying out the detecting step of themethod of the invention, an aliquot of the PCR reaction (typically 1 μl)is added to 9 μl of formamide HI-DI and 0.25 μl of GeneScan marker −500LIZ size standard. After denaturation, the sample is placed in the ABI3130 Genetic Analyzer and capillary electrophoresis is carried out. Theraw data is analysed using GeneScan software. This analysis is veryimportant since the PCR products will be sized by extrapolation to anin-sample size standard. Using this technique inventors are able todetect in a very precise and accurate manner the mutation of interest.

The invention is further illustrated with the following Examples, whichis provided to illustrate certain embodiments of the present inventionand is not to be construed as limiting the invention.

EXAMPLE 1 Determination of the ELREA Deletion at the Exon 19 of the EGFRGene by GeneScan Materials and Methods:

1—Sample Collection

Venous blood (10 ml) from each subject was placed into tubes containing50 mmol of EDTA (ethylenediaminetetraacetic acid) per liter, and genomicDNA was isolated with the QIAmp® DNA blood Mini kit (Qiagen, Germany),according to manufacturer's instructions.

2—GeneScan Reaction Preparation

1.1—Materials

-   -   Abi Prism 3130 DNA Analyser (Perkin-Elmer, Applied Biosystems)    -   96 Well optical Reaction plate (Applied Biosystems, Cat. No,        4306737)

1.2—PCR reaction mix with PNA probe

The PCR reaction mix was made as follows:

-   -   2.5 μl Buffer 10× (Ecogen)    -   b 0.5 μl 50 mM MgCl₂ (Ecogen)    -   0.625 μl 10 mM dNTPs (Promega)    -   1,25 [2l 10 μM of each primer    -   0.1 μl TAQ polymerase (Ecogen)    -   12.5 μl 5 mM PNA probe (Applied Biosystems)    -   5 μl DNA from serum or plasma    -   Add sterile distilled H₂0 to a final volume of 25 μl.

1.3—PCR reaction mix without PNA probe

The PCR reaction mix was made as follows:

-   -   2.5 μl Buffer 10× (Ecogen)    -   0.5 μl 50 mM MgCl₂ (Ecogen)    -   0.625 μl 10 mM dNTPs (Promega)    -   1.25 μl 10 μM of each primer    -   0.1 μl TAQ polymerase (Ecogen)    -   5 μl DNA from serum or plasma    -   Add sterile distilled H₂0 to a final volume of 25 μl.        The reverse primer was labelled with the 6-FAM fluorophore        (6-FAM emits fluorescence with a peak wavelength of 522 nm).

Primer Forward: (SEQ ID NO: 1) ACTCTGGATCCCAGAAGGTGAG Primer Reverse:(SEQ ID NO: 2) 6-FAM- CCACACAGCAAAGCAGAAACTC PNA probe sequence:(SEQ ID NO: 3) Ac-AGATGTTGCTTCTCTTA

1.3—PCR program

The PCR was performed as follows: 95° C. during 5 minutes followed by 40cycles at 95° C. for 30 seconds, 58° C. for 30 seconds and 72° C. for 1minute, and a final extension of 72° C. for 5 minutes.

3—GeneScan Preparation

-   -   9 μl formamide HI-DI (Applied Biosystems)    -   0.25 μl GeneScan marker-500 LIZ size standard (Applied        Biosystems)    -   1 μl final PCR product diluted

Samples were denaturated at 93° C. for 3 minutes and cooled on ice for10 minutes. They were then subjected to capillary electrophoresis andsubsequent subjected to an excitation wavelength of 494 nm for detectionof emission wavelength at 522 nm on the ABI 3130 DNA analyzer.

Results:

A total of 41 serum/plasma samples were analysed by GeneScan fordetection of detections in exon 19. All samples analysed were frompatients with positive mutations in tumor tissue.

GeneScan analysis results show that 55% of the samples with positivemutations in tumor tissue were also positive by Genescan analysis (Table1).

TABLE 1 Exon 19 EGFR mutation analysis in serum/plasma of patients withpositive mutations in tumor tissue. EGFR mutations in tumor S/P positiveS/P negative N = 41 N = 22 (55%) N = 19 (45%) Male 8 8 Female 14 11Erlotinib 1st-line 12 11 2nd-line 10 8 Never smoker 15 12 Ex-smoker 7 6Smoker 0 1 Blood extraction Before treatment 16 9 After treatment 5 8Unknown 1 2 CR + PR 1 + 12 1 + 6 SD + PD 0 + 1  2 + 0

EXAMPLE 2 Determination of the L858R Mutation in Exon 21 of the EGFRGene by Genescan Materials and Methods:

1—Sample Collection

-   -   Venous blood (10 ml) from each subject was placed into tubes        containing 50 mmol of EDTA (ethylenediaminetetraacetic acid) per        liter, and genomic DNA was isolated with the QIAmp® DNA blood        Mini kit (Qiagen, Germany), according to manufacturer's        instructions.

2—Taqman Reaction (5′Nuclease Activity Assay)

1.1—Materials

-   -   AB 7000 or 7900HT (Applied Biosystems) 1.2—PCR reaction        (5′Nuclease activity assay) mix with PNA probe

The PCR reaction mix was made as follows (Table 2):

TABLE 2 The PCR was performed as follows: 60° C. durante 2 min followedby 50 cycles at 95° C. during 10 min followed by 50 cycles at 95° C. for15 seconds and 60° C. during 1 min 30 sec. Final Stock For each ReactionMix Concentr Concentr sample (μl) Universal TaqMan Master Mix 1x 2x 12.5Primer F 0.6 μM 10 μM 1.5 Primer R 0.6 μM 10 μM 1.5 Probe wt-VIC 0.2 μM10 μM 0.5 Probe mut-FAM 0.2 μM 10 μM 0.5 PNA 0.5 μM 10 μM 1.25 Serum orplasma DNA 5 Water 5.25

3—Primers, Probes and PNA

The probe for detecting wild-type sequences was labeled with thefluorochrome VIC, whereas the probe for detecting the mutant allele waslabeled with the fluorochrome FAM

Primer Forward: (SEQ ID NO: 4) AACACCGCAGCATGTCAAGA Primer Reverse:(SEQ ID NO: 5) TTCTCTTCCGCACCCAGC

Probe for the detection of the wild-type allele labelled with thefluorochrome VIC:

(SEQ ID NO: 6) VIC-TCACAGATTTTGGGCTGGCCAAAC-TAMRA

Probe for the detection of the mutant allele labelled with thefluorochrome FAM:

(SEQ ID NO: 7) 6-FAM-CAGATTTTGGGCGGGCCAAAC-TAMRA (SEQ ID NO: 8)PNA Probe: AGTTTGGCCAGCCCA

Results:

A total of 41 serum/plasma samples were analysed by GeneScan fordetection of mutations in L858R. All samples analysed were from patientswith positive mutations in tumour tissue.

5′ nuclease activity assay analysis results show that 55% (L858R) of thesamples with positive mutations in tumour tissue were also positive bythis analysis (Table 3).

TABLE 3 EGFR mutations in tumor S/P positive S/P negative N = 41 N = 22(55%) N = 19 (45%)

EXAMPLE 3 Determination of the T790M Mutations in Exon 21 of the EGFRGene by Taqman Reacion Materials and Methods: 1—Sample Collection

Venous blood (10 ml) from each subject was placed into tubes containing50 mmol of EDTA (ethylenediaminetetraacetic acid) per liter, and genomicDNA was isolated with the QIAmp® DNA blood Mini kit (Qiagen, Germany),according to manufacturer's instructions.

2—Taqman Reaction (5′Nuclease Activity Assay)

1.1—Materials

-   -   AB 7000 or 7900HT (Applied Biosystems)

1.2—PCR reaction (5′ Nuclease activity assay) mix with PNA probe

The PCR reaction mix was made as in example 2 (see Table 2):

The PCR was performed as follows: 60^(a)C for 2 min followed by 50cycles at 95° C. during 10 min followed by 50 cycles at 95° C. for 15seconds and 60° C. during 1 min 30 sec.

3—Primers, Probes and PNA

The probe for detecting wild-type sequences was labeled with thefluorochrome VIC, whereas the probe for detecting the mutant allele waslabelled with the fluorochrome FAM

Primer Forward: (SEQ ID NO: 9) AGGCAGCCGAAGGGC Primer Reverse:(SEQ ID NO: 10) CCTCACCTCCACCGTGCA

Probe for the detection of the wild-type allele labelled with thefluorochrome VIC:

(SEQ ID NO: 11) VIC- TGAGCTGCGTGATGA-MGB

Probe for the detection of the mutant allele labelled with thefluorochrome FAM:

(SEQ ID NO: 12) 6-FAM-TGAGCTGCATGATGA-MGB (SEQ ID NO: 13)PNA Probe: TCATCACGCAGCTC

Results:

A total of 4 serum/plasma samples were analysed. All samples analysedwere from patients with positive mutations in tumour tissue.

5′Nuclease activity assay analysis results show that 75% (T790M) of thesamples with positive mutations in tumour tissue were also positive bythis analysis (Table 4 for T790M).

TABLE 4 EGFR mutations in tumor S/P positive S/P negative N = 4 N = 3(75%) N = 1 (25%)

1. A method for the detection of mutations at the EGFR gene in a serumor plasma sample from a subject, said method comprising: (i) obtainingDNA from said sample; (ii) amplifying a nucleic acid sequencecorresponding to a specific target region of the EGFR gene comprising amutation to be detected by means of PCR using primers capable ofamplifying said target region of the EGFR gene and a Protein-NucleicAcid probe wherein said Protein-Nucleic Acid probe is capable ofspecifically recognizing and hybridizing with the EGFR wild-typesequence within said specific region of the EGFR gene thereby inhibitingthe amplification of the wild-type sequence and producing PCR productsfrom the gene comprising the mutation to be detected; and (iii)detecting the PCR products obtained in (ii) whereby the detection of thePCR product indicates that there is a mutation in the EGFR gene.
 2. Themethod according to claim 1, wherein the mutation at nucleic acidsequence corresponding to a specific region of the EGFR gene to bedetected comprises a mutation is-selected from the group consisting of:ELREA deletions at the exon 19, an L858R mutation at the exon 21, and aT790M mutation at the exon
 20. 3. The method according to claim 1,wherein the detecting is carried out by means of nucleic acidsequencing.
 4. The method according to claim 1, wherein theamplification is carried out using a primer labeled with one detectablemoiety and wherein the detecting step is carried out by means ofcapillary electrophoresis.
 5. The method according to claim 4, whereinsaid the primer labeled with one detectable moiety is an oligonucleotideprimer which is fluorescently labelled at its 5′ end with a fluorescentdye.
 6. The method of claim 5, wherein said fluorescent dye is selectedfrom the group consisting of 6-FAM, HEX and NED dyes.
 7. The method ofclaim 1, wherein the detection of the PCR product is carried out using a5′ nuclease activity assay.